Profiling of Endogenous Peptides in Human Synovial Fluid by NanoLC−MS:  Method Validation and Peptide Identification

Abstract

Synovial fluid potentially contains markers for early diagnosis and disease progression in degenerative joint diseases such as osteoarthritis. Here, a method is described for profiling endogenous peptides in human synovial fluid, using ultrafiltration, solid-phase extraction, nanoscale liquid chromatography, and high-resolution mass spectrometry. Synovial fluid is characterized by its high viscosity, caused by the presence of the lubricant hyaluronic acid. The method proved to be capable of eliminating the high concentrations of hyaluronic acid, which appeared to be necessary to obtain satisfactory analytical performance, that is, within-day relative standard deviations of 5-15%, between-day relative standard deviations of 6-16%, a linear response of R2=0.994, a limit of detection in the femtomole range, and reproducible recoveries of 14-67%. With the developed method, in a synovial fluid sample from an osteoarthritis patient and a healthy control, in total, 501 peptides originating from 40 proteins were identified. Peptide cleavage products from six proteins that have been associated with osteoarthritis in earlier studies (collagen II, proteoglcycan 4, serum amyloid A, tubulin, vimentin, and Matrix Gla) could also be identified with our profiling method. The robustness of the method indicates that it can be applied in systems biology approaches for further studies on degenerative joint disease, eventually leading to a better understanding of the disease and its therapy, as well as the development of novel biomarkers to monitor these processes.

Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity

“…aggrecan, heparin sulfate, and cartilage oligomeric matrix protein) (55,56). Lubricin is a mucin-like glycoprotein.…”

Orbitrap mass spectrometry: Instrumentation, ion motion and applications

“…The high resolving power (>150,000) and excellent mass accuracy (specified as $2-5 ppm, but demonstrated to be as low as 0.2 ppm under favorable conditions) (Makarov et al, 2006a,b), significantly reduce false positive peptide identifications in bottom-up protein analyses (Adachi et al, 2006;Charneau et al, 2007;Dave et al, 2007;de Souza, Godoy, & Mann, 2006;Forner, Arriaga, & Mann, 2006;Graumann et al, 2008;Hanke et al, 2008;Hoopmann, Finney, & MacCoss, 2007;Kamphorst et al, 2007;Li et al, 2007b;Lu et al, 2007Lu et al, , 2008Manes et al, 2008;Mayampurath et al, 2008;Park et al, 2008;Scherl et al, 2008;Scigelova & Makarov, 2006;Shi et al, 2007;Swatkoski et al, 2007Swatkoski et al, , 2008Thevis et al, 2007b;Usaite et al, 2008;Venable et al, 2007;Wilson-Grady, Villén, & Gygi, 2008;Wisniewski et al, 2007;Zougman et al, 2008), and improve the accuracy of de novo interpretations of MS/MS spectra (DiMaggio et al, 2008;Ford et al, 2008;Frank et al, 2007;Scigelova & Makarov, 2006;Yates et al, 2006). The LTQOrbitrap has also been used to analyze intact small (3-10 kDa) (Bredehöft, Schänzer, & Thevis, 2008;Thevis et al, 2006a;Thevis, Thomas, & Schänzer, 2008b), medium (10-25 kDa) (Macek et al, 2006;…”

Glycoproteomics of Lubricin-Implication of Important Biological Glyco- and Peptide-Epitopes in Synovial Fluid

“…Or does lubricin fragmentation patterns in OA or RA differ from those in healthy individuals? Recent studies reported the fragment of lubricin in SF (Gobezie et al, 2007;Kamphorst et al, 2007). In the work of Kamphorst et al, they found two lubricin C-terminal fragments (R 1285 PALNYPVYGETTQV 1299 and D 1373 QYYNIDVPSRTA 1385 ) in OA SF but not in healthy SF (Kamphorst et al, 2007).…”

“…Recent studies reported the fragment of lubricin in SF (Gobezie et al, 2007;Kamphorst et al, 2007). In the work of Kamphorst et al, they found two lubricin C-terminal fragments (R 1285 PALNYPVYGETTQV 1299 and D 1373 QYYNIDVPSRTA 1385 ) in OA SF but not in healthy SF (Kamphorst et al, 2007). In the current study with enriched RA synovial lubricin, the first sequence (1285-1300) was found distributed throughout the gel; while the second sequence (1373-1385) was only detected in area 3 and 5, areas lower than the lubricin area ( Fig.…”

“…Though several proteomic analyses using synovial fluid samples have been carried out (Ruiz-Romero & Blanco, 2010), lubricin (or its fragments) appeared in only a few reports (Gobezie et al, 2007;Kamphorst et al, 2007;Estrella et al, 2010). To fully characterize synovial lubricin, the enriched samples were also subjected to proteomic analysis.…”